Duane P. Moser

Archaeal Diversity in Waters from Deep South African Gold Mines

ABSTRACT A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage ofEuryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated.

Environmental genomics reveals a single-species ecosystem deep within earth

DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earths crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

Alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep South African gold mine.

A novel extreme alkaliphile was isolated from a mine water containment dam at 3.2 km below land surface in an ultra-deep gold mine near Carletonville, South Africa. The cells of this bacterium were straight to slightly curved rods, motile by flagella and formed endospores. Growth was observed over the temperature range 20-50 degrees C (optimum 40 degrees C; 45 min doubling time) and pH range 8.5-12.5 (optimum pH 10.0). The novel isolate, one of the most alkaliphilic micro-organisms yet described, was a strictly anaerobic chemo-organotroph capable of utilizing proteinaceous substrates such as yeast extract, peptone, tryptone and casein. Elemental sulfur, thiosulfate or fumarate, when included as accessory electron acceptors, improved growth. The G+C content of genomic DNA was 36.4 mol %. Phylogenetic analysis based on the 16S rDNA sequence indicated that the isolate is a member of cluster XI within the low G+C gram-positive bacteria, but only distantly related to previously described members. On the basis of physiological and molecular properties, the isolate represents a novel species, for which the name Alkaliphilus transvaalensis gen. nov., sp. nov. is proposed (type strain SAGM1T = JCM 10712T = ATCC 700919T). The mechanism of generation of the highly alkaline microbial habitat and the possible source of the alkaliphile are discussed.

Desulfotomaculum and Methanobacterium spp. dominate a 4-to 5-kilometer-deep fault

ABSTRACT Alkaline, sulfidic, 54 to 60°C, 4 to 53 million-year-old meteoric water emanating from a borehole intersecting quartzite-hosted fractures >3.3 km beneath the surface supported a microbial community dominated by a bacterial species affiliated with Desulfotomaculum spp. and an archaeal species related to Methanobacterium spp. The geochemical homogeneity over the 650-m length of the borehole, the lack of dividing cells, and the absence of these microorganisms in mine service water support an indigenous origin for the microbial community. The coexistence of these two microorganisms is consistent with a limiting flux of inorganic carbon and SO42− in the presence of high pH, high concentrations of H2 and CH4, and minimal free energy for autotrophic methanogenesis. Sulfide isotopic compositions were highly enriched, consistent with microbial SO42− reduction under hydrologic isolation. An analogous microbial couple and similar abiogenic gas chemistry have been reported recently for hydrothermal carbonate vents of the Lost City near the Mid-Atlantic Ridge (D. S. Kelly et al., Science 307:1428-1434, 2005), suggesting that these features may be common to deep subsurface habitats (continental and marine) bearing this geochemical signature. The geochemical setting and microbial communities described here are notably different from microbial ecosystems reported for shallower continental subsurface environments.

Dating ultra-deep mine waters with noble gases and 36Cl, Witwatersrand Basin, South Africa

Abstract Concentrations and isotopic ratios of dissolved noble gases, 36Cl, δD and δ18O in water samples from the ultra-deep gold mines (0.718 to 3.3 km below the surface) in the Witwatersrand Basin, South Africa, were investigated to quantify the dynamics of these ultra deep crustal fluids. The mining activity has a significant impact on the concentrations of dissolved gases, as the associated pressure release causes the degassing of the fissure water. The observed under saturation of the atmospheric noble gases in the fissure water samples (70–98%, normalized to ASW at 20°C and 1013 mbar) is reproduced by a model that considers diffusive degassing and solubility equilibration with a gas phase at sampling temperature. Corrections for degassing result in 4He concentrations as high as 1.55 · 10−1cm3STP4He g−1, 40Ar/36Ar ranging between 806 and 10331, and 134Xe/132Xe and 136Xe/132Xe ratios above 0.46 and 0.44, respectively. Corrected 134(136)Xe/132Xe and 134(136)Xe/4He-ratios are consistent with their production ratios, whereas the nucleogenic 4He/40Ar, and 134(136)Xe/40Ar ratios generally indicate that these gases are produced in an environment with an average [U + Th]/K-content 2–3 times above that of crustal average. In two scenarios, one considering only accumulation of in situ produced noble gases, the other additionally crustal flux components, the model ages for 14 individual water samples range from 13 to 168 Ma and from 1 to 23 Ma, respectively. The low 36Cl-ratios of (4–37) · 10−15 and comparatively high 36Cl-concentrations of (8–350) · 10−15 atoms 36Cl l−1 reflect subsurface production in secular equilibrium indicating an age in excess of 1.5 Ma or 5 times the half-life of 36Cl. In combination, the results suggest residence times of the fluids in fissures in this region (up to 3.3 km depth) are of the order of 1–100 Ma. We cannot exclude the possibility of mixing and that small quantities of younger water have been mixed with the very old bulk.

The Distribution of Microbial Taxa in the Subsurface Water of the Kalahari Shield, South Africa

Microbial communities within deep subsurface environments were analyzed by 16S rRNA gene cloning. Clone libraries from 27 borehole fluid, 7 mining-contaminated, and 5 rock samples were compared. Borehole fluids derived from deep fractures were populated by microbial communities with low diversity with an average of 11 and 5 bacterial and archaeal OTUs respectively. Low taxa richness was likely driven by limited biogeochemical reactions available for growth and not extreme parameters such as pH and temperature. Novel taxa of Firmicutes were discovered, commonly found in warm, slightly alkaline, anoxic fracture fluids. Highly divergent lineages of Archaea, unique to South African deep subsurface fracture fluids, are also described. Clone library clustering analyses based on LIBSHUFF phylogenetic relatedness revealed distinct groups of samples corresponding with sample source and geochemistry.

Temporal Shifts in the Geochemistry and Microbial Community Structure of an Ultradeep Mine Borehole Following Isolation

A borehole draining a water-bearing dyke fracture at 3.2-km depth in a South African Au mine was isolated from the open mine environment. Geochemical, stable isotopic, nucleic acid-based, and phospholipid fatty acid (PLFA) analyses were employed as culture-independent means for assessing shifts in the microbial community and habitat as the system equilibrated with the native rock-water environment. Over a two-month period, the pH increased from 5.5 to 7.4, concurrent with a drop in pe from −2 to −3. Whereas rDNAs related to Desulfotomaculum spp. represented the major clone type encountered throughout, lipid biomarker profiling along with 16S rDNA clone library and terminal restriction fragment length polymorphism (T-RFLP) analyses indicated the emergence of other Gram-positive and deeply-branching lineages in samples during the later stages of the equilibration period. A biofilm that formed on the mine wall below the borehole produced abundant rDNAs related to the α Proteobacteria. β- and γ −Proteobacteria appeared to transiently bloom in the borehole shortly after isolation. Chemical modeling and sulfur isotope analyses of the borehole effluent indicated that microbial sulfate reduction was the major terminal electron-accepting process shortly after isolation, whereas Fe+3 reduction dominated towards the end of the experiment. The persistence of Desulfotomaculum-like bacteria throughout suggests that these organisms adapted to changing geochemical conditions as the redox decreased and pH increased following the isolation of the borehole from the mine atmosphere. The restoration of anaerobic aquatic chemistry to this borehole environment may have allowed microbiota indigenous to the local basalt aquifer to become more dominant among the diverse collection of bacterial lineages present in the borehole.

The origin and age of biogeochemical trends in deep fracture water of the Witwatersrand Basin, South Africa

Water residing within crustal fractures encountered during mining at depths greater than 500 meters in the Witwatersrand basin of South Africa represents a mixture of paleo-meteoric water and 2.0–2.3 Ga hydrothermal fluid. The hydrothermal fluid is highly saline, contains abiogenic CH 4 and hydrocarbon, occasionally N 2 , originally formed at ∼ 250–300°C and during cooling isotopically exchanged O and H with minerals and accrued H 2 , 4 He and other radiogenic gases. The paleo-meteoric water ranges in age from ∼ 10 Ka to > 1.5 Ma, is of low salinity, falls along the global meteoric water line (GMWL) and is CO 2 and atmospheric noble gas-rich. The hydrothermal fluid, which should be completely sterile, has probably been mixing with paleo-meteoric water for at least the past ∼100 Myr, a process which inoculates previously sterile environments at depths > 2.0 to 2.5 km. Free energy flux calculations suggest that sulfate reduction is the dominant electron acceptor microbial process for the high salinity fracture water and that it is 10 7 times that normally required for cell maintenance in lab cultures. Flux calculations also indicate that the potential bioavailable chemical energy increases with salinity, but because the fluence of bioavailable C, N and P also increase with salinity, the environment remains energy-limited. The 4 He concentrations and theoretical calculations indicate that the H 2 that is sustaining the subsurface microbial communities (e.g. H 2 -utilizing SRB and methanogens) is produced by water radiolysis at a rate of ∼1 nM yr −1 . Microbial CH 4 mixes with abiogenic CH 4 to produce the observed isotopic signatures and indicates that the rate of methanogenesis diminishes with depth from ∼ 100 at < 1 kmbls, to < 0.01 nM yr −1 at > 3 kmbls. Microbial Fe(III) reduction is limited due to the elevated pH. The δ13C of dissolved inorganic carbon is consistent with heterotrophy rather than autotrophy dominating the deeper, more saline environments. One potential source of the organic carbon may be microfilms present on the mineral surfaces.

Shewanella pealeana sp. nov., a member of the microbial community associated with the accessory nidamental gland of the squid Loligo pealei.

A new, mesophillic, facultatively anaerobic, psychrotolerant bacterium, strain ANG-SQ1T (T = type strain), was isolated from a microbial community colonizing the accessory nidamental gland of the squid Loligo pealei. It was selected from the community on the basis of its ability to reduce elemental sulfur. The cells are motile, Gram-negative rods (2.0-3.0 microns long, 0.4-0.6 micron wide). ANG-SQ1T grows optimally over the temperature range of 25-30 degrees C and a pH range of 6.5-7.5 degrees C in media containing 0.5 M NaCl. 16S rRNA sequence analysis revealed that this organism belongs to the gamma-3 subclass of the Proteobacteria. The closest relative of ANG-SQ1T is Shewanella gelidimarina, with a 16S rRNA sequence similarity of 97.0%. Growth occurs with glucose, lactate, acetate, pyruvate, glutamate, citrate, succinate, Casamino acids, yeast extract or peptone as sole energy source under aerobic conditions. The isolate grows anaerobically by the reduction of iron, manganese, nitrate, fumarate, trimethylamine-N-oxide, thiosulfate or elemental sulfur as terminal electron acceptor with lactate. Growth of ANG-SQ1T was enhanced by the addition of choline chloride to growth media lacking Casamino acids. The addition of leucine or valine also enhanced growth in minimal growth media supplemented with choline. The results of both phenotypic and genetic characterization indicate that ANG-SQ1T is a Shewanella species. Thus it is proposed that this new isolate be assigned to the genus Shewanella and that it should be named Shewanella pealeana sp. nov., in recognition of its association with L. pealei.

Geochemically Generated, Energy-Rich Substrates and Indigenous Microorganisms in Deep, Ancient Groundwater

Recent studies have shown that the biosphere extends to depths that exceed 3 km, raising questions regarding the age of the microbes in these deep ecosystems and their sources of energy for metabolism. Abiogenic energy sources that are derived from in situ, purely geochemical sources and thus independent from photosynthesis have been suggested. We sampled saline fracture water emanating from a 3.1-km deep borehole in a Au mine in the Witwatersrand Basin of South Africa and characterized the chemical constituents (including stable isotopes), groundwater age, and indigenous microorganisms. Salinity data and ratios of dissolved noble gases indicate that extremely ancient (2.0 Ga) saline fracture water has mixed with meteoric water to yield an average subsurface residence time of 20–160 Ma, the oldest age of any waters collected to date in the Witwatersrand Basin. H2 isotope data suggest the water originated from a depth of 4 to 5 km. Sulfur isotope fractionation indicates biological sulfate reduction. Calculations of free energies and steady state energy fluxes based on water chemistry data also support sulfate reduction as the dominant terminal electron accepting process. Lipid and flow cytometry data indicate a sparse microbial community (103 cells ml−1), despite the presence of relatively high concentrations of energy-rich compounds (H2, CH4, CO, ethane, propane, butane, and acetate). The H2 can be explained by radiolysis of water. Stable isotopic signatures of the CH4 and short chain hydrocarbons indicate abiogenic synthesis. The persistence of energy-rich compounds suggests that other factors are limiting to microbial metabolism and growth, e.g., availability of an inorganic nutrient, such as Fe or phosphate.